This invention generally relates to a method and regulatory compound for desensitization of receptors. In particular, the invention relates to a method and regulatory compound for desensitization of B cell receptors, Fe receptors and NK receptors. The invention also relates to compounds and methods for sensitization of receptors.
The B cell antigen receptor complex is composed of membrane immunoglobulin noncovalently associated with heterodimers of Ig-xcex1 and Ig-xcex2. These signal transducing subunits contain a conserved ITAM motif(immunoreceptortyrosine-based activation motif) required for signal transduction (Cambier, 1995). Aggregation of the BCR by multivalent antigen initiates transphosphorylation of the Ig-xcex1 and Ig-xcex2 ITAM motifs and activation of receptor-associated kinases (for review see DeFranco, 1997; Kim et al., 1993; Kurosaki, 1997). Phosphorylated ITAMs recruit additional effectors such as PI3-K, PLC-xcex3 and members of the Ras/MAPK pathway. These signaling events are responsible for B cell proliferation, and increased expression of activation markers such as MHC class II and CD86, that are required to prime the B cell for subsequent interactions with Th cells.
The B cell repertoire is finely tuned to contain maximal receptor diversity in the absence of autoreactivity. Autoreactive clones are eliminated by processes including clonal deletion by apoptosis or receptor editing, and anergy (Goodnow et al., 1988; Hartley et al., 1993; Hertz and Nemazee, 1997; Nemazee and Burki, 1989; Rathmnell et al., 1996). In the latter case, autospecific cells persist but are unresponsive to antigen. The molecular mechanisms underlying B cell unresponsiveness have been studied in BCR transgenic mice and in several in vitro models of receptor desensitization (Brunswick et al., 1994; Cambier et al., 1988; Cambier et al., 1990, Erikson et al., 1991; Gay et al., 1993; Nemazee and Burki, 1989; Okamoto et al., 1992; Vilen et al., 1997). Studies in the HEL/anti-HEL double transgenic mouse have shown that B cells tolerant to selfantigen exhibit reduced cell surface expression of IgM, are no longer capable of antigen-induced CD86 expression, and are sensitive to Fas mediated apoptosis (Goodnow etal., 1989; Ho et al., 1994; Rathmell et al., 1996). In a number of these models, receptor desensitization is characterized by the inability of antigen to elicit tyrosine phosphorylation or renewed Ca2+ mobilization despite the continued expression of antigen binding receptors.
Recently, disruption of receptor proximal signaling events have been studied in desensitized B cells (Cooke et al., 1994; Vilen et al., 1997). Results from these studies reveal a lack of antigen induced phosphorylation and activation of receptor associated kinases such as Lyn, Blk, and Syk. Johnson et al. showed that receptor-associated kinases could be activated by exposure to doubly phosphorylated ITAM peptides, suggesting that the failure of desensitized receptors to activate signaling pathways was not due to a defect intrinsic to the kinase, but rather reflected a defect at the level of the receptor and its ability to couple to Lyn (Johnson et al., 1995). Johnson et al. hypothesize that receptor unresponsiveness may be due to an uncoupling of Lyn from an otherwise intact mIg/Igxcex1xcex2 complex, or alternatively, a result of excessive phosphotyrosine phosphatase activities at the receptor.
Despite considerable research in this area, previous investigators have failed to teach or suggest the molecular event that the present inventors have shown to be responsible for maintaining the unresponsive phenotype of desensitized receptors. Therefore, prior to the present invention, therapeutic compounds which specifically target this molecular event have not been identified.
A wide variety of medical treatments require regulation of the immune response in a patient. Such treatments include, for example, vaccinations, treatments for autoimmune diseases, immunodeficiency diseases, immunoproliferative diseases, and treatments involving the transplantation of organs and skin. Traditional reagents and methods used to regulate a subject""s immune response often results in unwanted side effects. For example, immunosuppressive reagents such as cyclosporin A, azathioprine, and prednisone are used to suppress the immune system of a patient with an autoimmune disease or patients receiving transplants. Such reagents, however, suppress a patient""s entire immune response, thereby crippling the ability of the patient to mount an immune response against infectious agents not involved in the original disease. Due to such harmful side effects and the medical importance of immune regulation, reagents and methods to regulate specific parts of the immune system have been the subject of study for many years.
The present invention can be used to overcome traditional problems with immunoregulatory reagents by targeting specific cells and immune receptors in vivo.
The present invention generally relates to methods and compounds for desensitizing a receptor selected from the group consisting of a B cell antigen receptor (BCR), a pro-B cell receptor (pro-BCR), a pre-B cell receptor (pre-BCR), immunoglobulin Fc receptor (FcR) and natural killer (NK) cell receptor. Such a method includes the step of contacting a compound with such a receptor that has an extracellular ligand binding component and a transducer component, wherein contact with the compound: (1) causes a dissociation of the extracellular ligand binding component from the transducer component when the two components are associated prior to contact with the compound, and/or (2) inhibits association of the extracellular ligand binding component with the transducer component when the two components are dissociated prior to contact with the compound, thereby desensitizing the receptor.
The present invention also relates to a method and compounds for sensitizing or prolonging/enhancing sensitization of a receptor selected from the group consisting of BCR, pro-BCR, pre-BCR, FcR and NK receptor. Such a method includes the step of contacting a compound with such a receptor that has an extracellular ligand binding component and a transducer component, wherein the compound: (1) causes the extracellular ligand binding component to associate with the transducer component when the two components are not associated with each other prior to contact by the compound; and/or (2) prolongs or enhances the time over which the extracellular ligand binding component is associated with the transducer component when the components are associated prior to contact by the compound, thereby sensitizing the receptor.
More particularly, one embodiment of the present invention relates to a method to desensitize a B cell antigen receptor, and preferably, by selectively desensitizing a B cell antigen receptor which binds to a specific antigen. Such a method is useful for treating any B cell-related disorder in which desensitization of the B cell antigen receptor would provide a therapeutic benefit, alone or in conjunction with another treatment. Such a method is also useful for research and diagnostic assays, and for screening putative regulatory compounds. Such method includes the step of contacting a regulatory compound with a B cell antigen receptor that has an mIg component and a transducer component including Igxcex1 and/or Igxcex2, wherein contact with the compound: (1) causes a dissociation of the mIg component from the transducer component when the two components are associated prior to contact with the compound, and/or (2) inhibits association of the mIg component with the transducer component when the two components are dissociated prior to contact with the compound, thereby desensitizing the B cell antigen receptor. The mIg component can be either IgD or IgM. A B cell-disorder that can be treated by the present method can include, but is not limited to, autoimmune disease (e.g., rheumatoid arthritis or systemic lupus erythematosus), malignancies and transplantation. A particularly preferred disorder to treat with the method of the present invention is an autoimmune disease. Preferably, the compound selectively targets a BCR having a particular antigen specificity, such as a B cell antigen receptor which specifically binds to an autoantigen. Such a method is advantageous in that functions of normal or desirable B cells can be left intact, while functions of abnormal or undesirable B cells can be inhibited.
Another embodiment of the present invention relates to a method to sensitize or prolong/enhance sensitization of a BCR. Such a method is useful, for example, for increasing or inducing a B cell response to a given antigen or antigens, and can be used in a vaccine or adjuvant system. In one embodiment, the method includes the step of contacting a compound with a B cell antigen receptor that has an mIg component and a transducer component including Igxcex1 and Igxcex2, wherein the compound: (1) causes the mIg component to associate with the transducer component when the components are not associated with each other prior to contact by the compound; and/or (2) prolongs or enhances the time over which the mIg component is associated with the transducer component when the components are associated prior to contact by the compound, thereby enhancing sensitization of the B cell antigen receptor for treatment of the disorder. In one embodiment of this method, an additional factor can be contacted with the B cell antigen receptor, such as an antigen or other factor which enhances vaccination against a given antigen or enhancement of the B cell antigen response.
One embodiment of the present invention relates to a method to treat autoimmune disease, including the step of contacting a compound with an autoreactive B cell antigen receptor that has an mIg component associated with a transducer component including Igxcex1 and Igxcex2, wherein contact with the compound: (1) causes a dissociation of the mIg component from the transducer component when the two components are associated prior to contact with the compound, and/or (2) inhibits association of the mIg component with the transducer component when the two components are dissociated prior to contact with the compound, thereby desensitizing the B cell antigen receptor for treatment of an autoimmune disease.
Another embodiment of the present invention relates to a method desensitize an Ig Fc receptor (FcR). Such a method is useful for treating any disorder in which desensitization of an FcR, and particularly, a specific FcR, would provide a therapeutic benefit, alone or in conjunction with another treatment. Such a method is also useful for research and diagnostic assays, and for screening putative regulatory compounds. Such disorders include, but are not limited to, an allergic disorder; and disorders related to inflammatory responses including antibody-dependent cell-mediated cytotoxicity, release of inflammatory mediators and regulation of antibody production, and more particularly include, but are not limited to, thrombocytopenia purpura, rheumatoid arthritis, systemic lupus erythematosus, type II and type III hypersensitivity reactions, and allergic inflammation. The method includes the step of contacting a compound with an Fc receptor that has an xcex1 receptor component and a transducer component, wherein contact with the compound: (1) causes a dissociation of the xcex1 receptor component from the transducer component when the two components are associated prior to contact with the compound, and/or (2) inhibits association of the xcex1 receptor component with the transducer component when the two components are dissociated prior to contact with the compound, thereby desensitizing the Fc receptor.
Another embodiment of the present invention relates to a method to treat allergic disorders, including the step of contacting a compound with an FCxcex5RI receptor that has an xcex1 receptor component and a transducer xcex2/xcex3 component, wherein contact with the compound: (1) causes a dissociation of the xcex1 receptor component from the transducer component when the two components are associated prior to contact with the compound, and/or (2) inhibits association of the xcex1 receptor component with the transducer component when the two components are dissociated prior to contact with the compound, thereby desensitizing the Fcxcex5RI receptor for treatment of the allergic disorder.
Another embodiment of the present invention relates to a method to identify compounds useful for desensitizing a receptor, including the steps of: (1) providing an assay system including a receptor selected from a B cell antigen receptor, a pro-B cell receptor, a pre-B cell receptor or an Fc receptor, wherein the receptor includes an extracellular ligand binding component and at least one transducer component, and wherein the extracellular ligand binding component is associated with the transducer component; (2) contacting the receptor with a compound to be evaluated; and, (3) determining whether the compound, when contacted with the receptor, is capable of causing the extracellular ligand binding component to dissociate from the transducer component. In the case of a B cell antigen receptor, the extracellular ligand binding component is mIg and the transducer components are Igxcex1 and Igxcex2. In the case of an FcR receptor, the extracellular ligand binding receptor is typically the xcex1 receptor chain, and the transducer component varies depending on the specific FcR, as is known in the art. For the Fcxcex5RI, the transducer components are xcex2 and xcex3 chains.
Yet another embodiment of the present invention relates to a method to identify compounds useful for desensitizing a receptor, including the steps of: (1) providing an assay system including a receptor selected from a B cell antigen receptor or an Fc receptor, wherein the receptor includes an extracellular ligand binding component and at least one transducer component, and wherein the extracellular ligand binding component is not associated with the transducer component; (2) contacting the receptor with a compound to be evaluated; and, (3) determining whether the compound, when contacted with the receptor, is capable of inhibiting the extracellular ligand binding component from associating with the transducer component.
The above methods can include cell-based assays and non-cell based assays. Compounds identified by the above methods are useful for treating a variety of B cell and FcR-associated disorders, including autoimmune disease and allergic disorders. The methods of desensitizing/sensitizing a receptor and compounds useful in such methods can be performed/used in vivo, in vitro, and/or ex vivo. In addition, the above-described regulatory compounds, including those identified by the present method, can be used in a therapeutic composition and in a method of treatment or desensitization of the present invention.
Yet another embodiment of the present invention relates to a compound useful for treating a condition selected from the group consisting of a B cell-associated disorder and an FcR-associated disorder. Such a compound is characterized by its ability to: (1) cause a dissociation of an extracellular ligand binding component from a transducer component in a B cell antigen receptor or an Fc receptor when the two components are associated prior to contact with the compound, and/or (2) inhibit association of the extracellular ligand binding component with the transducer component when the two components are dissociated prior to contact with the compound, thereby desensitizing the receptor. In a preferred embodiment, the compound is selected from an antibody, a peptide, or a mimetope thereof. In one embodiment, the compound is an antibody. Such an antibody can be a monovalent antibody, a divalent antibody, or a bi-specific antibody.
Another embodiment of the present invention relates to an isolated antibody which selectively binds to a BCR, wherein the antibody, upon binding to such a BCR having an mIg component associated with Igxcex1 and Igxcex2 components, is capable of inducing the mIg component to dissociate from the Igxcex1 and Igxcex2 components, thereby desensitizing the BCR. In one embodiment, the BCR is expressed by a B cell. Such an antibody can be used in a method of the present invention to desensitize a B cell antigen receptor.
Another embodiment of the present invention relates to an isolated antibody which selectively binds to an FcR, wherein the antibody, upon binding to such an FcR that has an xcex1 receptor component associated with at least one transducer component, is capable of inducing the xcex1 receptor component to dissociate from the transducer component, thereby desensitizing the FcR. In one embodiment, the FcR is Fcxcex5RI, and the transducer components are xcex2 and xcex3 chains. In another embodiment, the FCxcex5RI is expressed by a mast cell, a basophil or an eosinophil. Such an antibody can be used in a method of the present invention to desensitize an Fc receptor.
All patents and publications referenced herein are incorporated by reference in their entireties.